*2.2. Patients*

This study was conducted at Kurume University Hospital between September 2011 and May 2016. A total of 105 subjects, including 41 patients with UC, 34 with CD, and 30 healthy subjects were enrolled. Prior to commencing the study, 19 subjects were excluded due to an insu fficient sample volume. The patient diagnosis was based on characteristic clinical, endoscopic, radiological, and histological features. The patient characteristics and the medical therapy they received are summarized in Table 1.

### *2.3. Clinical Evaluations*

For the evaluation of disease activity, clinical activity in patients with UC was graded using the partial Mayo score (PMS), with the inactive disease defined as a score ≤2, with no individual sub-score >1 point [16]. Patients with CD were graded according to the CD activity index (CDAI) comprised of eight factors, each added after adjustment with a weighted factor, with the inactive disease defined as a score <150 points [17].


#### **Table 1.** Patient characteristics.

IQR: Interquartile range.

#### *2.4. Determination of Laboratory Parameters*

Blood samples were collected from all patients and were used to measure the following laboratory parameters: Total leukocyte count, serum levels of hemoglobin, albumin, and C-reactive protein (CRP).

#### *2.5. Separation of PBMCs and RNA Extraction*

Blood samples (10 mL) were obtained by cubital venous puncture and collected in standard sterile polystyrene vacuum tubes with heparin. First, freshly drawn blood was diluted at a ratio of 1:2.5 with a phosphate bu ffered saline. PBMCs were isolated from the diluted blood by a Ficoll-Paque (GE Healthcare, Uppsala, Sweden) density gradient centrifugation according to the manufacturer's instructions. PBMCs were pelleted, snap-frozen on dry ice, and stored at −80 ◦C until use [18]. RNA was extracted from PBMC samples following the protocol described for the TRIzol reagen<sup>t</sup> (Invitrogen, Carlsbad, CA, USA). The quantity and purity of the RNA were determined for all samples on a Nanodrop ND-1000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). The average yield was 23,000 ng. The purity, as measured by the A260/280 ratio, was between 1.91 and 1.95 [18].

#### *2.6. Measurement of TRP Channel mRNA Expression Using Real-Time Quantitative Polymerase Chain Reaction (Real-Time qPCR)*

Total RNA was converted into cDNA using the ReverTra Ace qPCR RT kit (Toyobo, Osaka, Japan). The generated cDNAs (25 ng) were stored at −20 ◦C. cDNA was added to the TaqMan Gene Expression Master Mix (Applied Biosystems, Foster City, CA, USA). qPCR reactions (20 μL) composed of 2 μL cDNA template, TaqMan Universal PCR Master Mix (2<sup>×</sup>, Thermo Fisher Scientific, Foster City, CA, USA), TaqMan assay (20<sup>×</sup>, Thermo Fisher Scientific), and H2O. RT-PCR was performed using the StepOne Real-Time PCR System (Applied Biosystems). Reactions, run in triplicate, were incubated at 50 ◦C for 2 min and 95 ◦C for 10 min, followed by 40 cycles of 95 ◦C for 15 s and 60 ◦C for 1 min. The TaqMan probe and primer sets for the target genes used in this study are shown in Table 2.


**Table 2.** Details of TaqMan probes and primers used in this study.

GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; TRP: Transient receptor potential.

GAPDH was used as the reference gene. Ct values for GAPDH mRNA of an individual PBMC per sample were calculated. The mean was calculated from experiments performed in duplicate. For data analysis, the StepOne software v2.1 was used. Data representing the relative expression of detected mRNA normalized to GAPDH mRNA was used as a calibrator for comparative analysis. RT-qPCR was performed in accordance with the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines [19,20]. The relative expression data was calculated according to the 2-ΔΔCt method.

### *2.7. Statistical Analyses*

Results were analyzed using the JMP v12 statistical package (SAS Institute, Cary, NC, USA). The normality of distribution was assessed using the Shapiro–Wilk test. As mRNA levels of each TRP

channel were all not normally distributed, statistical analyses were performed using Mann–Whitney U and Kruskal–Wallis H tests and correlation analysis was performed using Spearman's rank correlation test. The Bonferroni-corrected Mann–Whitney U test was used to evaluate inter-group comparisons of the mean differences according to their distribution. Data are shown as the mean ± standard deviation (SD) or as correlation coefficients.
